Vascular endothelial growth factor receptors: Molecular mechanisms of activation and therapeutic potentials

Experimental Eye Research 83 (2006) 1005e1016 www.elsevier.com/locate/yexer

Review

Vascular endothelial growth factor receptors: Molecular mechanisms of activation and therapeutic potentials Nader Rahimi* Departments of Ophthalmology and Biochemistry, School of Medicine, Boston University, Boston, MA 02118, USA Received 10 February 2006; accepted in revised form 24 March 2006 Available online 19 May 2006

Abstract Angiogenesis-associated eye diseases are among the most common cause of blindness in the United States and worldwide. Recent advances in the development of angiogenesis-based therapies for treatment of angiogenesis-associated diseases have provided new hope in a wide variety of human diseases ranging from eye diseases to cancer. One group of growth factor receptors critically implicated in angiogenesis is vascular endothelial growth factor receptors (VEGFR), a subfamily of receptor tyrosine kinases (RTKs). VEGFR-1 and VEGFR-2 are closely related receptor tyrosine kinases and have both common and specific ligands. VEGFR-1 is a kinase-impaired RTK and its kinase activity is suppressed by a single amino acid substitution in its kinase domain and by its carboxyl terminus. VEGFR-2 is highly active kinase, stimulates a variety of signaling pathways and broad biological responses in endothelial cells. The mechanisms that govern VEGFR-2 activation, its ability to recruit signaling proteins and to undergo downregulation are highly regulated by phosphorylation activation loop tyrosines and its carboxyl terminus. Despite their differential potentials to undergo tyrosine phosphorylation and kinase activation, both VEGFR-1 and VEGFR-2 are required for normal embryonic development and pathological angiogenesis. VEGFR-1 regulates angiogenesis by mechanisms that involve ligand trapping, receptor homodimerization and heterodimerization. This review highlights recent insights into the mechanism of activation of VEGFR-1 and VEGFR-2, and focuses on the signaling pathways employed by VEGFR-1 and VEGFR-2 that regulate angiogenesis and their therapeutic potentials in angiogenesis-associated diseases. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: vascular endothelial growth factor receptor-1 (VEGFR-1); vascular endothelial growth factor receptor-2 (VEGFR-2); FLT-1; FLK-1; angiogenesis; vasculogenesis; receptor tyrosine kinases; Src kinases; phosphoinositide 3-kinase; PLC-gamma1; c-Cbl; ubiquitination; downregulation; signal transduction; tyrosine phosphorylation

1. Introduction VEGFR-1/FLT-1 (fms-like tyrosine kinase) and VEGFR-2/ KDR/FLK-1 (fetal liver kinase) are the prototypes of a gene family encoding structurally related receptors, FLT-3/FLK-2 and FLT-4/VEGFR-3 and belong to the receptor tyrosine kinase (RTK) subfamily (Hanks and Quinn, 1991; Blume-Jensen and Hunter, 2001). VEGFR-1 and VEGFR-2 are primarily involved in angiogenesis (Yancopoulos et al., 2000) where FLT-3 and FLT-4 are involved in hematopoiesis and lymphogenesis

* Tel.: þ1 617 638 5011; fax: þ1 617 638 5337. E-mail address: [email protected] 0014-4835/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2006.03.019

(Jussila and Alitalo, 2000). The diversification of the VEGFR family proteins during evolution, through the advent of multiple ligands and receptors has created a decisive signaling network capable of controlling angiogenesis. Through utilization of receptor homo- and heterodimers, activated by specific and common ligands, diverse angiogenic signals are propagated. An additional level of angiogenic signaling diversity is obtained through differential activation of distinct signaling molecules downstream of each receptor. VEGFR-1 is a kinase-impaired RTK, and may signal in the context of receptor heterodimer (Rahimi et al., 2000; Autiero et al., 2003; Neagoe et al., 2005). In contrast, VEGFR-2 is highly kinase active receptor and activates broad signaling cascades leading to diverse biological responses (Waltenberger et al.,

1006

N. Rahimi / Experimental Eye Research 83 (2006) 1005e1016

1994; Meyer and Rahimi, 2003; Rahimi, 2006). Three different gene products including, PLGF, VEGF-A, VEGF-B are known to bind VEGFR-1. VEGF-A, VEGF-D and VEGF-C are known to bind VEGFR-2 (Neufeld et al., 1999; Tammela et al., 2005). VEGF-C and VEGF-D also bind to VEGFR-3, an RTK that is expressed by lymphatic endothelial cells and hematopoietic progenitor cells (Jussila and Alitalo, 2000; Tammela et al., 2005). VEGFR-1 and VEGFR-2 are structurally similar, consisting of an extracellular ligand-binding domain with a seven immunoglobulin (Ig)-like motif, a single transmembrane domain, a juxtamembrane domain, a kinase domain split by a kinase insert, and a carboxyl terminus. Overall, there is 43.2% sequence homology between VEGFR-1 and VEGFR-2. The extracellular domain of VEGFR-1 and VEGFR-2 displays 33.3% homology and the cytoplasmic region 54.6%. The kinase domains of VEGFR-1 and VEGFR-2 represent the most conserved region with 70.1% homology. In contrast, the carboxyl terminus represents the most divergent region with only 28.1% sequence homology (Fig. 1).

2. VEGFR-1 and angiogenesis Initial evidence linking VEGFR-1 to endothelial cell function and angiogenesis was provided by targeted deletion of VEGFR-1, which resulted in early embryonic lethality due to abnormal blood vessel growth (Fong et al., 1995). Although the precise role of VEGFR-1 in endothelial cell functions is still emerging, in recent years several lines of evidence have developed that suggest VEGFR-1 may play both negative and positive roles in angiogenesis. The negative role of VEGFR-1 in angiogenesis was suggested based on the observation that loss of VEGFR-1/flt-1 in mice caused an increase in the number of endothelial progenitors, resulting in the vascular disorganization (Fong et al., 1995, 1999). How VEGFR-1 contributes to angiogenesis in a negative manner is not fully clear. Some studies have suggested that the negative role of VEGFR1 in angiogenesis is associated with its ability to alter endothelial cell division (Kearney et al., 2002, 2004). However, work from several laboratories indicates that selective activation of VEGFR-1, either by creating VEGFR-1 chimeras (Rahimi et al., 2000; Zeng et al., 2001) or VEGF mutants is not associated with proliferation of endothelial cells in vitro (Keyt, 1996). In these studies ligand stimulation of VEGFR-1 induced neither cell proliferation nor apoptosis. Selective activation of VEGFR-1 also did not promote cell migration or intracellular calcium release (Meyer et al., 2004a, 2004b). It could be argued that in a defined condition when VEGFR-1 is activated by a ligand that only allows receptor homodimerization and with no cross-talk with other RTKs such as VEGFR-2 and VEGFR-3, VEGFR-1 is not capable of promoting biological responses such as endothelial cell migration, cell proliferation and intercellular calcium release. In line with this notion, in a recent gene targeting study where the entire cytoplasmic region of VEGFR-1 including its kinase domain was deleted, the knockin truncated VEGFR-1 mice developed with no aberrant vasculogenesis (Hiratsuka et al.,

1998). This was in startling contrast to the VEGFR-1 knockout mice, which mice died in early development due to overgrowth of endothelial cells (Fong et al., 1995, 1999). In sum, these observations reinforce the idea that VEGFR-1 may act as a decoy receptor and its function in angiogenesis may involve its ligand biding extracellular region, acting as a VEGF-trap to modulate VEGFR-2 function (Fig. 2). It should be noted that role of VEGFR-1 in angiogenesis is far more complex than initially thought. Clearly, the ability of VEGFR-1 to modulate angiogenesis is not limited to a mechanism in which it acts as a VEGF-trapping receptor. For example, stimulation of VEGFR-1 with PLGF, (a VEGFR-1 specific ligand) promotes heterodimerization of VEGFR-1 with VEGFR-2 leading to transactivation of VEGFR-2 and angiogenesis (Autiero et al., 2003). Overexpression of PLGF, in transgenic mice is also reported to promote angiogenesis (Odorisio et al., 2002). These findings suggest that VEGFR-1 when paired with VEGFR-2 via a mechanism of heterodimerization its activity positively regulates angiogenesis. Also, it is increasingly apparent that activation of VEGFR-1 in endothelial cells and its final biological function is subject to the microenvironment of endothelial cells, in particular, the presence or absence of the other VEGFR family proteins and neuropilins (Klagsbrun et al., 2002). Likewise, VEGFR-1 homodimerization and heterodimerization may define the inhibitory or stimulatory nature of its signaling relays (Rahimi et al., 2000; Zeng et al., 2001; Autiero et al., 2003). Another factor that may determine the nature of VEGFR-1 signaling in the endothelium microenvironment is the type of ligand being utilized to activate VEGFR-1. The nature of the signal induced by PLGF, which selectively binds VEGFR-1, might be different than that induced by VEGF-A and VEGFB. In agreement with this idea, the impaired angiogenesis associated with plgf null mice was not rescued by VEGF-B (Carmelite, 2003). Also, a naturally occurring VEGF-PLGF heterodimer may favor the formation of certain heterodimerization complexes among the VEGFR family involving VEGFR-1, VEGFR-2 and neuropilins, which may activate a unique signaling pathway that might not be possible by stimulation with either VEGF or PLGF alone. It should also be noted that endothelial cells are morphologically and genetically different from each other (Cleaver and Melton, 2003) raising the possibility that variation in endothelial cells may ultimately determine or modify the nature of VEGFR-1 signaling. Finally, there are also some evidences that suggest VEGFR-1 signaling in non-endothelial cells might be different that of the endothelial cells. For example, stimulation of VEGFR-1 with PLGF in non-endothelial cells such as monocytes and trophoblasts induce cell migration and proliferation (Clauss et al., 1996; Athanassiades and Lala, 1998; Dikov et al., 2005). VEGFR-1 activity also is linked to pathological angiogenesis such as cancer and retinopathy of prematurity. It has been reported that activation of VEGFR-2 prevents pathological regression of blood vessels from oxygen-induced retinal vascular degeneration in retinopathy of prematurity (Shih et al., 2003) and VEGFR-1 is also expressed by some carcinomas and is involved in cell migration and cell invasion (Wey et al., 2005).

N. Rahimi / Experimental Eye Research 83 (2006) 1005e1016

1007

43.2%

70.1%

33.3%

TM JM KM KI

KM

C-terminal

VEGF-B

PLGF

VEGFR-1 (FLT-1)

VEGF-A

VEGF-C

Extracellular domain

28.1%

VEGFR-2 (FLK-1/KDR)

VEGF-D

54.6%

1050 VEGFR-1

D F G L AR D I Y K

N

PD

Y

V RKGDTR LPLK WMA

P E SI

VEGFR-2

D F G LA R D I

Y K

D

PD

Y

V RKGDAR LPLK WM A

P E TI

PDGFR-β PDGFR-α

D F G LA R D I

MR

D

SN

Y

I SKGSTF LP LK WM A

P E SI

D F G LA R D I

MH

D

SN

Y

V SKGSTFLPV K WMA

P E SI

CSF-1R

D F G LA R D I

MN

D

SN

Y

I VKGNARLPVK WM A

P E SI

c-Kit

D F G LA R D I

KN

D

SN

Y

VV KGNAR LP K WM A

P E SI

FLK-2

D F G LA R D I

LS

D

SS

Y

VVRGNARLPVK WM A

P E SL

FLT-4

D F G LA R D I

YK

D

PD

Y

V RKGSARLPLK WM A

P E SI

Activation loop

1050 Human

DFGLARDIYK N PDYVRKG D TRLPLKWMAPES I...

Mouse

DFGLARDIYK N PDYVRRG D TRLPLKWMAPES I…

Rat

DFGLARDIYK N PDYVRRG D TRLPLKWMAPES I…

Chicken

DFGLARDIYK N PDYVRKG D ARLPLKWMAPES I…

Fig. 1. Schematic representation of VEGFR-1, VEGFR-2 and their corresponding ligands: multiple VEGF ligands bind to VEGFR-1 and VEGFR-2. Placenta growth factor (PLGF) and VEGF-B bind to VEGFR-1 but not to VEGFR-2. VEGF-C and VEGF-D bind VEGFR-2 but not to VEGFR-1. VEGF-A binds to both VEGFR-1 and VEGFR-2. VEGFR-1 and VEGFR-2 are structurally similar, consisting of an extracellular ligand-binding domain with seven immunoglobulin (Ig)-like motifs, a single transmembrane domain (TM) and juxtamembrane (JM) domain, a kinase domain (KD) split by a kinase insert (KI), and a carboxyl terminus. The sequence homology between VEGFR-1 and VEGFR-2 also is shown. The kinase domain of VEGFR-1 and VEGFR-2 represent the most conserved region with 70.1% homology. The carboxyl terminus represents the most divergent region with only 28.1% sequence homology. Sequence alignment of the catalytic and activation loop regions of human VEGFR-1 and various receptor tyrosine kinases of type III family are shown. The residue corresponding to asparagine (N) 1050 of VEGFR-1 is aspartic acid (D) in type III kinases. The location asparagine (N) 1050 in the activation loop of human VEGFR-1 is indicated by arrow. This residue is highly conserved and is invariable among the type III family kinases except VEGFR-1. Asparagine 1050 of VEGFR-1 is conserved among human (GenBank accession number: NM002019), mouse (GenBank accession number: BAA24498) rat (GenBank accession number: P53767) and chicken (GenBank accession number: BAB84690).

3. VEGFR-1 is a kinase-impaired RTK The mechanisms by which most RTKs are activated are well characterized. Transphosphorylation of RTK and their ability to phosphorylate target proteins are the hallmark of RTK activation. In this context, VEGFR-1 is considered to

be a kinase-impaired RTK (e.g., VEGFR-1 is poorly tyrosine phosphorylated and its ability to phosphorylate a polypeptide substrate is negligible). All RTKs, including VEGFR-1 contain an evolutionary conserved kinase domain containing GXGXXG, an ATP binding site, HRDLA, a motif essential for catalysis and one or two tyrosine autophosphorylation sites

1008

N. Rahimi / Experimental Eye Research 83 (2006) 1005e1016

VEGF-trapping (decoy model) VEGFR-1 VEGFR-1

Ligand

Receptor heterodimerization

VEGFR-1

VEGFR-2

Receptor homodimerization

VEGFR-1 VEGFR-1

Ligand Ligand

Activation of signaling proteins

Modulation of angiogenesis

Fig. 2. Schematic representation of the mechanisms of involvement of VEGFR-1 in angiogenesis: VEGF-trapping: the mechanism by which VEGFR-1 binds to VEGF and prevents it from binding to VEGFR-2. The cytoplasmic domain of VEGFR-1 is not required for its VEGF-trapping function (Hiratsuka et al., 1998). VEGFR-1 may elicit its role in angiogenesis by heterodimerization with VEGFR-2 and VEGFR-3 (Rahimi et al., 2000; Autiero et al., 2003; Dixelius et al., 2003). The third mechanism by which VEGFR-1 regulates angiogenesis is through receptor homodimerization. This type of activation of VEGFR-1 leads to activation of signaling proteins that might regulate angiogenesis in either positive or negative manner (Rahimi et al., 2000; Meyer et al., 2004a, 2004b).

(Hanks and Quinn, 1991). Despite having these motifs, the ligand stimulation of VEGFR-1 results only in minor tyrosine phosphorylation of VEGFR-1 in vivo and in vitro (de Vries et al., 1992; Waltenberger et al., 1994; Rahimi et al., 2000). In some RTKs such as colon carcinoma kinase-4 (CCK4), ErbB3/HER3, KLG, Ror1 and DRL abnormalities in the kinase domain have been suggested to contribute to the kinase-defective phenotype of these RTKs (Chou and Hayman, 1991; Guy et al., 1994; Mossie et al., 1995; Yoshikawa et al., 2001). VEGFR-1 has no aberrant amino acid replacement in the GXGXXG and HRDLA motifs. A recent study suggests that the kinase-impaired activity of VEGFR-1 can be rescued, in part, by swapping the carboxyl terminus of VEGFR-1 with that of VEGFR-2. The carboxylterminus swapped VEGFR-1 promoted ligand-dependent autophosphorylation of VEGFR-1 and induction of endothelial cell proliferation (Meyer et al., 2004a, 2004b). It appears that this effect is selectively associated with its carboxyl terminus because replacement of the juxtamembrane (JM) region of VEGFR-1 with that of VEGFR-2 does not rescue its kinaseimpaired characteristic (Gille et al., 2000). A detailed understanding of the molecular mechanism underlying the carboxyl

terminus of VEGFR-1 in its poor tyrosine phosphorylation should wait until the crystal structure of VEGFR-1 is resolved. To date, the crystal structure of VEGFR-1 is not available, only its ligand-binding region is resolved (Wiesmann et al., 1997). How the carboxyl terminus of VEGFR-2 is able to rescue the kinase-impaired activity of VEGFR-1 is not fully understood. However, it is feasible that the carboxyl terminus of VEGFR-1 somewhat obstructs ligand-mediated autophosphorylation of VEGFR-1. In some RTKs, such as Tie-2, deletion of carboxyl terminus has been shown to enhance its autophosphorylation and kinase activation, suggesting that the carboxyl terminus negatively regulates tyrosine autophosphorylation possibly by interacting with activation loop and keeping it in an inhibitory conformation (Niu et al., 2002). The molecular explanation for the kinase-impaired characteristic of VEGFR-1 is provided by a recent finding that the decoy characteristic of VEGFR-1 is linked to the replacement of a highly conserved amino acid residue, located in its activation loop (Meyer et al., 2006). This amino acid is highly conserved among all the type III RTKs and corresponds to aspartic acid (D) but in VEGFR-1 is substituted to asparagine (N) (Fig. 1). Mutation of asparagine (N1050) within the

N. Rahimi / Experimental Eye Research 83 (2006) 1005e1016

activation loop to aspartic acid promoted enhanced liganddependent tyrosine autophosphorylation and kinase activation in vivo and in vitro. This mutation promotes endothelial cell proliferation, but not tubulogenesis. It also displayed an oncogenic phenotype as its expression in fibroblast cells elicited transformation and colony growth. It has been suggested that the conserved aspartic acid in the activation loop favors the transphosphorylation of the activation loop tyrosines and its absence renders VEGFR-1 to a less potent enzyme perhaps by disfavoring transphosphorylation of activation loop tyrosines. It should be emphasized that although these observations may explain the decoy characteristic of VEGFR-1 (e.g., impaired kinase activation and tyrosine phosphorylation), nevertheless these findings still do not explain mechanisms by which VEGFR-1 contributes to angiogenesis as a kinase-impaired RTK. Several RTK including, DRL, CCK4, KLG, Ror1 and ErbB3 all display impaired kinase activity, yet they transduce signals and regulate variety of biological functions (Chou and Hayman, 1991; Guy et al., 1994; Mossie et al., 1995; Yoshikawa et al., 2001). The best-studied kinase-impaired RTK is ErbB3, one of the four members of EGFR family (Citri et al., 2003). Data from several laboratories using various systems revealed that signaling of ErbB3 requires kinase activity of another EGFR family including ErbB1, ErbB2 and ErbB4 (Pinkas-Kramarski et al., 1996; Citri et al., 2003). DRL, another kinase-impaired RTK, although it shows no kinase activity in vivo and in vitro, nevertheless its activity is required for axon guidance in Drosophila (Yoshikawa et al., 2001). The molecular mechanism of DRL signaling and whether DRL transduces signal like ErbB3 is not known. There is some circumstantial evidence suggesting that VEGFR-1 may signal through other VEGFR family kinases, in particular, through VEGFR-2. Both VEGFR-1 and VEGFR-2 are expressed by endothelial cells, suggesting that under a favorable condition such as availability of appropriate ligand, VEGFR-1 may form heterodimer complexes with the other receptors. Indeed, VEGFR-1 has been shown to heterodimerize with VEGFR-2 and that heterodimerization leads to autophosphorylation, activation of VEGFR-2, and angiogenesis (Autiero et al., 2003). Thus, the mechanism by which VEGFR-1 supports angiogenesis is complex and likely involves several different mechanisms including VEGF-trapping, activation by heterodimerization and homodimerization (Fig. 2). VEGFR-1 also has a residual kinase activity and its selective activation that results only in homodimerization leads to activation of a limited number of signaling proteins such as p42/44 MAPK (Meyer et al., 2004a, 2004b). Thus, the unique signaling nature of VEGFR-1 and mechanisms by which it stimulates biological responses indicates that we have more to learn about RTK signaling than the current widely accepted and generalized RTK paradigm would suggest. 4. VEGFR-2 activation is essential for angiogenesis The role of VEGFR-2 in angiogenesis is well established and more comprehensive reviews on the role of VEGFR-2 in

1009

angiogenesis have been recently published (Yancopoulos et al., 2000; Carmeliet, 2003; Jain, 2003). In this review we focus on the recent advancement on VEGFR-2 activation and its signal transduction relays. 4.1. Carboxyl terminus is critical for VEGFR-2 activation and its signaling The mechanisms by which most RTKs transduce signal have been studied extensively at the molecular level. The basic principles of RTKs activation can be summarized as following: ligand-mediated dimerization of receptor monomers, transphosphorylation by dimerized receptors and docking of signaling proteins to receptor phosphotyrosines (Hubbard et al., 1998; Blume-Jensen and Hunter, 2001). The autophosphorylation sites are either involved in the regulation of kinase activity of the receptor or serve as a binding site for SH2- and PTB-containing proteins (Blume-Jensen and Hunter, 2001; Pawson, 2004). Central to ligand-induced RTK activation is the phosphorylation of one or two tyrosine residues within the activation loop (Hubbard et al., 1998). In the unstimulated form, the activation loop orients these tyrosine sites toward the activation sites of the enzyme and thereby sterically prevents binding to ATP-Mg2þ. It is suggested that phosphorylation of these tyrosines in the catalytic loop orients the inhibitory loop away from the active site enabling the RTK to bind ATP-Mg2þ and efficiently phosphorylate substrates (Hubbard et al., 1998; Schlessinger, 2000). The recent crystal structure of VEGFR-2 by McTigue et al. (1999) showed that tyrosines 1054 and 1059, two highly conserved tyrosine residues located in the activation loop of VEGFR-2 are phosphorylated. An unexpected feature of the VEGFR-2 structure is the conformation of the activation loop. Although phosphorylated, the activation loop is disordered in the region that includes phosphotyrosine 1059. This is in contrast to the structures of other tyrosine kinases such as FGFR-1 and the insulin receptor, which are well ordered and participate in specific interactions (McTigue et al., 1999). In last few years some of the paradigms learned from studying other RTKs revealed that catalytic activity is regulated by other segments of RTKs. For instance, substitution of two tyrosine residues with phenylalanine in the juxtamembrane region of the PDGFR-b drastically reduces autophosphorylation and activation of this receptor (Mori et al., 1993). Similar observations were reported for the MuSK tyrosine kinase, a receptor tyrosine kinase for agrin, which is involved in the functioning of the neuromuscular synapse (Till et al., 2002) and EphB2, a receptor tyrosine kinase for ephrins (Wybenga-Groot et al., 2001; Schlessinger, 2003). Recent crystal structures of MuSK and EphB2 revealed that the juxtamembrane domains of these receptors are directly involved in the regulation of kinase activation. The data obtained from the crystal structures suggest that the juxtamembrane domain interacts with the kinase domain and represses catalytic activity (Schlessinger, 2003). Additional biochemical studies demonstrated that these tyrosines also bind to SH2-containing proteins (Wybenga-Groot et al., 2001). There are two tyrosine residues within the juxtamembrane of

1010

N. Rahimi / Experimental Eye Research 83 (2006) 1005e1016

VEGFR-2 (tyrosines 799 and 820 in mouse VEGFR-2). Replacement of these tyrosines to phenylalanine in VEGFR-2 does not impair the ligand-dependent activation of VEGFR-2 (Dayanir et al., 2001; Meyer et al., 2002). This suggests that phosphorylation of these tyrosines are not involved in the kinase activation of VEGFR-2. Unlike juxtamembrane tyrosine phosphorylation sites, however, phosphorylation of tyrosine 1212 (tyrosine 1214 in human VEGFR-2) in the carboxyl terminus plays an important role in autophosphorylation and kinase activation of VEGFR2. Mutation of tyrosine 1212 to phenylalanine impairs full activation of VEGFR-2 and its ability to efficiently activate signaling proteins. Mutation of the same tyrosine residue to glutamic acid, however, preserved its ability to undergo ligand-dependent autophosphorylation (Meyer et al., 2002). This suggests that tyrosine 1212 regulates VEGFR-2 autophosphorylation in a manner that is similar to that of tyrosine sites in the JM region of MuSK and EphB2. Surprisingly, this mutant receptor as recently reported preserved normal development and angiogenesis in knockin mice (Sakurai et al., 2005). This indicates that either the low level kinase activity of tyrosine 1212 mutant VEGFR-2 is sufficient to promote angiogenesis or that phosphorylation of tyrosine 1212 is not stringently required for function of VEGFR-2 in vivo. It is also conceivable that phosphorylation of other tyrosine sites in VEGFR-2 may compensate the lack of phosphorylation of tyrosine 1212. In agreement with this possibility, the mutant PDGFR-b lacking the binding sites for PLC-g1, PI 3 kinase, SHP2 and Ras-GAP has been shown to stimulate gene expression in fibroblast cells almost as efficiently as the wild-type PDGFR-b (Fambrough et al., 1999). Similarly, activation of Src kinases by many laboratories has been shown to be important for PDGFR-b autophosphorylation and its biological function (Mori et al., 1993). Yet, PDGFR-b is fully functional in SYF cells, deficient for Src family kinases (Klinghoffer et al., 1999). This argues that RTK signaling induces activation of signaling proteins with an overlapping role in vivo and thus the role of individual docking sites may be compensated for by the presence of other docking sites. Further analysis of the carboxyl terminus of VEGFR-2 revealed that this region is engaged in VEGFR-2 activation in multiple ways. First, the carboxyl terminus of VEGFR-2 contains several tyrosine phosphorylation sites including tyrosine 1212, 1173 and 1221. Tyrosines 1212 and 1221 are phosphorylated but the identity of signaling proteins that are recruited to these sites is not known (Meyer et al., 2002; and our unpublished data). As discussed earlier, the presence of tyrosine 1212 is required for full autophosphorylation of VEGFR-2 (Meyer et al., 2002). Tyrosine 1173 is phosphorylated in vivo and recruits p85 of PI3 kinase (Dayanir et al., 2001), PLCg1 (Takahashi et al., 2001; Meyer et al., 2003) and regulate phosphorylation of c-Cbl (our unpublished data). In addition to possessing multiple tyrosine phosphorylation sites, the carboxyl terminus of VEGFR-2 also contains putative serine phosphorylation sites. Mutation of serines 1188 and 1191 in the carboxyl terminus of VEGFR-2 is shown to impair the ligand-dependent down regulation of VEGFR-2 (Singh et al., 2005). This might

explain why deletion of carboxyl terminus impairs the VEGFR’s ability to undergo ligand-dependent downregulation (Meyer et al., 2004a, 2004b). Finally, deletion of the entire carboxyl terminus of VEGFR-2 is shown to impair its ligand-dependent autophosphorylation. It appears that this role of carboxyl terminus is independent of tyrosine 1212 and may involve residues other than tyrosines (Meyer et al., 2004a, 2004b). Altogether, the current literature provides a framework for understanding of regulation of VEGFR-2 activation and further demonstrates that the carboxyl terminus plays various important roles in VEGFR-2 functions ranging from its ability to regulate autophosphorylation and recruitment of signaling proteins to ligand-dependent downregulation. Future work to characterize function of the VEGFR-2 carboxyl terminus will require a detailed analysis including: identification of tyrosine and serine phosphorylation sites and signaling proteins that might interact with these sites. Finally, resolving the crystal structure of VEGFR-2 with its carboxyl terminus intact may provide further insight into regulation of this important protein. 4.2. Role of phosphoinositide 3-kinase in VEGFR-2 signaling and angiogenesis In the last few years, the phosphoinositide 3-kinase (PI3K) signal transduction pathway has emerged as one of the main signal routes that VEGFR-2 employs to stimulate endothelial cell survival and proliferation. PI3 kinase is a lipid kinase that converts the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3). Proteins with pleckstrin-homology (PH) domains, such as protein kinase B (PKB/Akt), phosphoinositide-dependent kinase-1 (PDK-1) and PDK-2, bind to PIP3. Akt is activated by PIP3, PDK1 and PDK2 leading to phosphorylation of a host of other proteins that affect cell proliferation, cell cycle progression and cell survival (Rameh and Cantley, 1999; Cantley, 2002). Various experiments using several in vivo and in vitro systems have demonstrated that activation of PI3 kinase by VEGFR-2 promotes endothelial cell survival, proliferation and angiogenesis (Fig. 3). First, Wortmannnin and LY294002, the PI3 kinase specific inhibitors and a dominant negative form of Akt, a downstream target of PI3 kinase, all are able to inhibit VEGF-stimulated endothelial cell survival (Gerber et al., 1998). Second, inhibition of PI3 kinase by overexpression of a dominant negative form of p85 of PI3 kinase inhibits endothelial cell proliferation and induction of c-fos (Thakker et al., 1999). Similarly, overexpression of PTEN, a lipid phosphatase that reverses PI3 kinase function by dephosphorylating its phospholipid products, inhibits VEGF-mediated endothelial cell proliferation. Overexpression of phosphatase inactive form of PTEN enhances VEGF-mediated endothelial cell proliferation (Huang and Kontos, 2002). Third, expression of a viral form of PI3 kinase and its target Akt promotes angiogenesis and VEGF expression in chicken embryo (Jiang et al., 2000). The most recent study on PI3 kinase also suggests that it may regulate angiogenesis by regulating expression of Tie-2 receptor (Lelievre et al., 2005).

N. Rahimi / Experimental Eye Research 83 (2006) 1005e1016

1011

VEGFR-2

pY799

PIP3

PIP2

pY949

PI3K

IP3

pY1006

DAG

PLCγ1 AKT

pY1173

VRAP S6 kinase

Ca 2+

Shb

PKC

c-Cbl

Cell proliferation

Cell Migration

Cell survival

Src

?

Tubulogenesis/ Angiogenesis

Permeability

Fig. 3. Schematic representation of VEGFR-2 mediated signal transduction and their role in endothelial biology: VEGFR-2 activation induces tyrosine phosphorylation and activation PLCg1 via tyrosines 1006 and 1173. Activation of PLCg1 leads to tubulogenesis and proliferation of endothelial cells. Activation of PI3 kinase by VEGFR-2 is mediated by tyrosines 799 and 1173 and is involved in proliferation and survival of endothelial cells. Tyrosine 1173 of VEGFR-2 also associates with the adaptor protein, Shb. Src family kinases and c-Cbl are also activated by VEGFR-2. Activation of Src is linked to endothelial cell survival and permeability (see text for details). Phosphorylation of tyrosine 1049 in the kinase insert region of VEGFR-2 binds to VRAP (Matsumoto et al., 2005).

A mechanistic explanation on how PI3 kinase is activated by VEGFR-2 is demonstrated by a chimeric VEGFR-2 system in which the PI3 kinase binding sites were mutated and its role in VEGFR-2-mediated endothelial cell proliferation was tested. The tyrosine mutant chimeric VEGFR-2 (where tyrosines 799 and 1173 of mouse VEGFR-2 were mutated) abolished the ability of VEGFR-2 to stimulate PI3 kinase activity and cell proliferation (Dayanir et al., 2001). Also, PI3 kinase-dependent proliferation of endothelial cells is linked to activation of S6 kinase. This is supported by the observation that a mutant chimeric receptor that fails to activate PI3 kinase also fails to activate S6 kinase (Dayanir et al., 2001). Consistent with this observation, selective inhibition of S6 kinase inhibits VEGF-induced endothelial cell proliferation and in vivo angiogenesis (Guba et al., 2002; Yamazaki et al., 2005). In agreement with a critical role of PI3 kinase activation in growth and survival of endothelial cells, a recent study also demonstrated that tumstatin, a naturally occurring angiogenesis inhibitor, promotes apoptosis of endothelial cells by inhibiting the PI3 kinase pathway (Sudhakar et al., 2003). 4.3. Role of phospholipase Cg1 in VEGFR-2 signaling and angiogenesis Phospholipase Cg1 (PLCg1) hydrolyzes phosphoinositides to generate the second messengers, inositol 1,4,5-trisphosphate

(IP3) and diacylglycerol (DAG). IP3 increases intracellular Ca2þ levels and DAG activates protein kinase C (PKC) leading to variety of cellular responses (Sekiya et al., 1999). Several lines of evidences indicate that activation of PLC-g1 plays a pivotal role in angiogenesis and VEGFR-2 signal transduction (Fig. 3). First, a genetic study shows that lack of PLCg1 in mice results in significantly diminished vasculogenesis and erythropoiesis (Liao et al., 2002). Second, in transgenic zebrafish the PLC-g1deficiency shows defects in arterial formation. The PLC-g1 deficient embryos also failed to respond to exogenous VEGF, highlighting the importance of PLC-g1 for VEGFR-2-mediated signal transduction relay (Lawson et al., 2003). However, the exact role of PLCg1 in angiogenesis is not clear. VEGFR-2-mediated activation of PLC-g1 in a certain endothelial cellular background is suggested to stimulate cell proliferation and in other endothelial cells to stimulate differentiation and tubulogenesis (Rahimi et al., 2000; Takahashi et al., 2001; Meyer et al., 2003). Phosphorylation of tyrosines 1006 and 1173 (Y1006 and 1173 correspond to Y1008 and 1175 in human VEGFR-2, respectively) are important for recruitment of PLCg1 by VEGFR-2 (Takahashi et al., 2001; Meyer et al., 2003). The tyrosine mutant VEGFR-2 that fails to activate PLCg1 also fails to stimulate tubulogenesis in vitro (Meyer et al., 2003). A recent knockin approach in mice showed that mutation of tyrosine 1173 of VEGFR-2 abrogates its ability to promote angiogenesis

1012

N. Rahimi / Experimental Eye Research 83 (2006) 1005e1016

(Sakkurai et al., 2004). Although this finding suggests an essential role for tyrosine 1173 in the regulation of angiogenesis, it is not clear whether the defect is exclusively linked with the inability of the tyrosine 1173 mutant VEGFR-2 to activate PLCg1. Tyrosine 1173 of VEGFR-2 is also known to recruit other signaling proteins such as p85 of PI3 kinase (Dayanir et al., 2001) and Shb (Holmqvist et al., 2004). 4.4. Role of Src kinases in VEGFR-2 signaling and angiogenesis Activation of Src kinases have been implicated in the regulation of a variety of biological responses including cell proliferation, migration, differentiation and cell survival (Bromann et al., 2004). Activation of many RTKs leads to stimulation of Src family kinase activity via tyrosine phosphorylated docking sites on RTKs and the SH2 domain of Src kinases (Boggon and Eck, 2004). Selective activation of VEGFR-2 stimulates Src phosphorylation in endothelial cells (Meyer et al., 2002). However, the precise mechanism involved is not established. In particular, Src binding sites on VEGFR-2 and biological importance of Src in VEGFR2-mediated signal transduction is not fully understood. A recent study demonstrated that Src kinase activity is required to protect endothelial cells from apoptosis and it appears that Src elicits its anti-apoptotic function via Raf-1 activation (Alavi et al., 2003). It has also been shown that the ability of VEGF to induce vascular permeability requires Src kinases. Curiously, angiogenesis is normal in mice deficient for Src and Yes (Eliceiri et al., 1999). Src activation also is implicated in GPCR (such as nucleotide receptor P2Y2)-mediated transactivation of VEGFR-2 but not in ligand-mediated VEGFR-2 activation (Meyer et al., 2002; Seye et al., 2004). Indeed, ligand-dependent autophosphorylation of VEGFR-2 is not compromised in SYF cells, deficient in Src, Yes and Fyn and also over-expression of a dominant negative form of Src in endothelial cells has no apparent effect on the ligand-stimulated VEGFR-2 autophosphorylation (Meyer et al., 2002). In addition to the role of c-Src in VEGFR-2-mediated signal transduction, Src also plays a key role in angiogenesis by promoting expression of VEGF in response to hypoxia (Mukhopadhyay et al., 1995). Further studies are required to delineate exact role of Src in VEGFR-2 signal transduction, in particular, its association with VEGFR-2 and tyrosine phosphorylation site on VEGFR-2 that might be involved in recruitment of Src to VEGFR-2. 4.5. Role of serine phosphorylation in VEGFR-2 signaling and angiogenesis In addition to activating positive signaling pathways, activation of RTKs also induces negative feedback signaling pathways that attenuate or antagonize positive signaling. One mechanism that attenuates RTKs signaling is a rapid depletion of the cellular receptor pool, a phenomenon termed downregulation (Soldi et al., 1999). Recent study indicates that VEGFR-2 undergoes ligand-dependent downregulation

and degradation in a novel mechanism involving non-classical PKCs. Singh et al. (2005) demonstrated that the carboxyl terminus is required for PKC-dependent downregulation of VEGFR-2. Surprisingly, the ligand-dependent downregulation and degradation of VEGFR-2 proceeds without involvement of its ectodomain and engagement of g-secretase activity. Mutation of serine sites at 1188 and 1191 compromised the ability of VEGFR-2 to undergo efficient ligand-dependent downregulation. These findings suggest that serines 1188 and 1191 of VEGFR-2 are phosphorylated either directly by PKC or by serine kinases whose activity is regulated by PKCs. Upon phosphorylation of these sites they may recruit an E3 ligase to VEGFR-2, leading to ubiquitinylation and degradation of VEGFR-2. The identity of the E3 ligase responsible for ubiquitinylation of VEGFR-2 is not known. It appears, however, that c-Cbl is not the E3 ligase that involved in ubiquitinylation and downregulation of VEGFR-2, although it is phosphorylated upon VEGFR-2 stimulation with ligand (Singh et al., 2005). Since c-Cbl is activated by VEGFR-2 it is interesting to know what role it plays in angiogenesis. 4.6. VEGF, VEGF receptors and ocular angiogenesis Vascular endothelial growth factor (VEGF) is normally expressed by many cell types of the retina, including retinal pigment epithelium (RPE) cells and ganglion cells (Miller et al., 1997; Ida et al., 2003). VEGF-A is the most potent angiogenic protein known and its expression is abnormally elevated in patients with age-related macular degeneration (AMD), diabetic retinopathy, and retinopathy of prematurity (Campochiaro et al., 1999; Witmer et al., 2003). Expression of VEGF-A and its receptors, VEGFR-1 and VEGFR-2 also play critical role during the embryonic development of retinal, choroidal vasculatures and neural retinal development (Robinson et al., 2001; Fruttiger, 2002). The survey of current literature indicates that VEGF not only plays a functional role during ocular development but also is involved in normal function of the retina and pathological conditions associated with it. Studies suggest that in Type I diabetes expression of VEGF is altered due to metabolic abnormalities including hyperglycemia, which represent major vascular complications of Type I diabetes (Chiarelli et al., 2000). Aberrant angiogenesis in diabetes is most clinically apparent in proliferative diabetic retinopathy. Hypoxia-induced expression of VEGF and other pro-angiogenic factors leads to altered angiogenesis, which causes major damage to pericytes. These aberrant conditions lead to increased permeability and rupture of retinal vessels (Caldwell et al., 2005). Although the role of angiogenesis is well documented in diabetic retinopathy, altered angiogenesis may also play a key role in diabetic neuropathy, diabetic nephropathy and diabetic wound healing (Isner et al., 2001; Kim et al., 2005). Furthermore, evidence now suggests that VEGF-A also promotes retinal vascular permeability and its presence is associated with the leakiness of retinal vessels in non-proliferative diabetic retinopathy (Murata et al., 1995). While the role of individual VEGF receptors involved in mediating vascular permeability

N. Rahimi / Experimental Eye Research 83 (2006) 1005e1016

is not fully understood, recent studies suggest that VEGFR-2 may play a prominent role in mediating the ability of VEGF to promote permeability of vascular endothelial cells (Joukov et al., 1998; Stacker et al., 1999). Elevated expression of VEGF-A due to hypoxia is considered to be the primary cause of retinopathy of prematurity (Smith, 2004). In addition, recent study suggests that PlGF, a VEGFR-1 specific ligand may protect against oxygen-induced vessel loss without stimulating angiogenesis (Shih et al., 2003). This finding implies that selective activation of VEGFR-1 but not VEGFR-2 may promote survival of retinal endothelial cells without promoting angiogenesis. It has been shown that VEGF-A plays a key role in choroidal angiogenesis, a major cause of visual loss in AMD. VEGF-A expression found to be significantly higher in patients with AMD (Zarbin, 2004) and its expression is also elevated in RPE cells in patients with AMD and in experimental animal models (Schwesinger et al., 2001). Altogether, the above-mentioned studies provide ample evidence for the central role of VEGF and its receptors in ocular angiogenesis. Further studies, however, are needed to delineate the exact role of VEGF-A and possible involvement of other VEGF family proteins in ocular angiogenesis. It is also essential to establish the relative contribution of VEGF receptors in the development of these conditions. Finally, it should be noted that signal transduction pathways activated by VEGF receptors in pathological angiogenesis, in particular, in ocular angiogenesis is largely unknown. Thus, it is critical to dissect the molecular mechanisms of signaling aspects of VEGF receptors in various pathological conditions and this certainly will be an important direction for future studies and ultimately new strategies for clinical application. 4.7. Ocular diseases and anti-angiogenesis therapy Although the idea of angiogenesis therapy was proposed over three decades ago (Folkman, 1971), the use of angiogenesis therapy was only realized in the last few years. Now, angiogenesis therapy is being considered for patients with cancer, age-related macular degeneration, heart diseases and others (Liu and Regillo, 2004; Annex and Simons, 2005; Neri and Bicknell, 2005; Wegewitz et al., 2005). The primary causes of vision loss in Western countries are macular degeneration and diabetic retinopathy. Although the mechanisms of each disease are distinct, they both involve abnormal angiogenesis. In addition, retinopathy of prematurity, a major cause of blindness in children, is associated with abnormal angiogenesis due to oxygen supplementation in the premature infants (Shih et al., 2003). In recent years several classes of anti-angiogenesis agents have been under development, many of these agents either target VEGF (e.g., VEGF antibodies) or VEGFRs (e.g., small molecule kinase inhibitors). Bevacizumab (Avastin; Genentech Inc., South San Francisco, CA) is a humanized monoclonal anti-VEGF-A antibody (Rosen, 2005). Avastin represents the first anti-angiogenesis drug that has recently been approved by the FDA for use in combination with standard chemotherapy in patients with advanced colorectal cancer. Another class of anti-angiogenesis

1013

inhibitors that are currently in clinical trails are the small molecule kinase inhibitors such as PTK787, SU11248 and ZD6474 which are potent inhibitors of VEGFR-1 and VEGFR-2 (Kerbel and Folkman, 2002; Rosen, 2005). To date, the anti-angiogenesis therapy for ocular diseases that have been evaluated are angiogenesis inhibitors that mainly bind to VEGF such as pegaptanib (Macugen), and VEGF-Trap (Saishin et al., 2003; Gragoudas et al., 2004; Zakarija and Soff, 2005). Pegaptanib and VEGF-Trap like Avastin bind to VEGF and prevent binding of VEGF to VEGF receptors. Survey of current anti-angiogenesis therapies indicate that these therapies may in some cases reduces disease progression but by and large they are ineffective in stopping or reversing the diseases that are being tested (Stacker et al., 1999; Kerbel and Folkman, 2002). Another challenge facing the application of these anti-angiogenesis therapies to ocular angiogenesis in diseases such as AMD is the requirement of repeated injection of these drugs into the patient’s eye. This represents a serious setback for usefulness of these drugs for patients with AMD. It appears that anti-angiogenesis therapy for ocular diseases may require a long-term administration of anti-angiogenesis drugs. Thus, preparation of these drugs in a sustained-release format may dramatically improve not only their efficacy but also the convenience of their application into the patients’ eye. In addition, other strategies such as gene therapy may overcome limitations of the aforementioned anti-angiogenesis drugs. Recent studies using viral vector-mediated delivery of anti-angiogenesis proteins in rodent models provide a new and possibly more effective strategy for treatment of ocular angiogenesis (Reich and Bennett, 2003). Thus, it is clear that further work on the efficacy and safety of these agents as well as the development of more selective, potent and safer anti-angiogenesis agents certainly will provide substantial advances for the treatment of angiogenesis-associated ocular diseases. One avenue toward the development of more selective and better anti-angiogenesis drugs is to target a specific signaling pathway of VEGF receptors.

5. Conclusions Abnormal angiogenesis is associated with pathology of many diseases including cancer, age-related macular degeneration (AMD), proliferative diabetic retinopathy (PDR), and retinopathy of prematurity (ROP). What is clear is that the more we learn about molecular mechanisms of VEGFR-1 and VEGFR-2 regulation and their signal transduction, the more we continue to learn about angiogenesis and molecules that regulate it. The emerging scenario in VEGFR-1 and VEGFR-2 activation is that the complexity of blood vessel development necessitated more stringent control over VEGFR-1. On one hand VEGFR-1 evolved to have a higher affinity to various ligands, on the other hand VEGFR-1 lost its potency in enzymatic activity, creating a unique mechanism to control its function. VEGFR-2 plays a broader role in angiogenesis; its activity regulates endothelial cell growth, differentiation, migration and tubulogenesis. The carboxyl terminus of

1014

N. Rahimi / Experimental Eye Research 83 (2006) 1005e1016

VEGFR-2 plays a central role in its activation, ability to recruit signaling proteins, to undergo downregulation and to promote angiogenesis. Finally it should be noted that activation and signaling of VEGF receptors appear to be regulated not only by each other in both negative and positive manners but also by other receptors including integrins (Soldi et al., 1999; Yeynolds et al., 2004), cadherins (Rahimi and Kazlauskas, 1999; Bussolino et al., 2001) and neuropilins (Klagsbrun et al., 2002). In addition, the fact that multiple VEGF ligands with different preference and capability bind to VEGFRs and activate them in homo- and heterodimeric manners presents a challenge to study their selective biochemical and cellular signaling in endothelial cell background. Although characterization of specific functions of VEGFR-1 and VEGFR-2 and their signal transduction relays has been a frustrating task, it has nevertheless been illuminating. Continued research into the function of VEGFRs as well as techniques involving creating chimeric VEGFRs in which the extracellular domain of each VEGFR is replaced with that of CSF-1R (Rahimi et al., 2000), and mutant VEGFs that selectively bind to VEGFRs (Keyt et al., 1996; Stacker et al., 1999) continue to provide insight into their specific roles in angiogenesis. Evaluation of targeted mouse knockouts (Fong et al., 1995) and knockin (Sakurai et al., 2005) and the use of RNA interference may provide further insight into the function of this important family of receptors.

Acknowledgements This work was supported in part through grants from The National Institutes of Health (NIH) EY0137061, EY012997 and a grant from Massachusetts Lions Foundation to N.R. I thank Amrik J. Singh and Rosana D. Meyer for their comments and reading the manuscript.

References Alavi, A., Hood, J.D., Frausto, R., Stupack, D.G., Cheresh, D.A., 2003. Role of Raf in vascular protection from distinct apoptotic stimuli. Science 301, 94e96. Annex, B.H., Simons, M., 2005. Growth factor-induced therapeutic angiogenesis in the heart: protein therapy (Review). Cardiovasc. Res. 65, 649e655. Athanassiades, A., Lala, P.K., 1998. Role of placenta growth factor (PIGF) in human extravillous trophoblast proliferation, migration and invasiveness. Placenta 19, 465e473. Autiero, M., Waltenberger, J., Communi, D., Kranz, A., Moons, L., Lambrechts, D., Kroll, J., Plaisance, S., De Mol, M., Bono, F., Kliche, S., Fellbrich, G., Ballmer-Hofer, K., Maglione, D., MayrBeyrle, U., Dewerchin, M., Dombrowski, S., Stanimirovic, D., Van Hummelen, P., Dehio, C., Hicklin, D.J., Persico, G., Herbert, J.M., Communi, D., Shibuya, M., Collen, D., Conway, E.M., Carmeliet, P., 2003. Role of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Flt1 and Flk1. Nat. Med. 9, 936e943. Blume-Jensen, P., Hunter, T., 2001. Oncogenic kinase signalling (Review). Nature 411, 355e365. Boggon, T.J., Eck, M.J., 2004. Structure and regulation of Src family kinases (Review). Oncogene 23, 7918e7927.

Bromann, P.A., Korkaya, H., Courtneidge, S.A., 2004. The interplay between Src family kinases and receptor tyrosine kinases (Review). Oncogene 23, 7957e7968. Bussolino, F., Serini, G., Mitola, S., Bazzoni, G., Dejana, E., 2001. Dynamic modules and heterogeneity of function: a lesson from tyrosine kinase receptors in endothelial cells (Review). EMBO Rep. 2, 763e767. Caldwell, R.B., Bartoli, M., Behzadian, M.A., El-Remessy, A.E., AlShabrawey, M., Platt, D.H., Liou, G.I., Caldwell, R.W., 2005. Vascular endothelial growth factor and diabetic retinopathy: role of oxidative stress. Curr. Drug Targets 6, 511e524. Campochiaro, P.A., Soloway, P., Ryan, S.J., Miller, J.W., 1999. The pathogenesis of choroidal neovascularization in patients with age-related macular degeneration (Review). Mol. Vis. 5, 34e38. Cantley, L.C., 2002. The phosphoinositide 3-kinase pathway (Review). Science 296, 1655e1657. Carmeliet, P., 2003. Angiogenesis in health and disease (Review). Nat. Med. 9, 653e660. Chiarelli, F., Spagnoli, A., Basciani, F., Tumini, S., Mezzetti, A., Cipollone, F., Cuccurullo, F., Morgese, G., Verrotti, A., 2000. Vascular endothelial growth factor (VEGF) in children, adolescents and young adults with Type 1 diabetes mellitus: relation to glycaemic control and microvascular complications. Diabetes Med. 17, 650e656. Chou, Y.H., Hayman, M.J., 1991. Characterization of a member of the immunoglobulin gene superfamily that possibly represents an additional class of growth factor receptor. Proc. Natl. Acad. Sci. USA 88, 4897e4901. Citri, A., Skaria, K.B., Yarden, Y., 2003. The deaf and the dumb: the biology of ErbB-2 and ErbB-3 (Review). Exp. Cell Res. 284, 54e65. Clauss, M., Weich, H., Breier, G., Knies, U., Rockl, W., Waltenberger, J., Risau, W., 1996. The vascular endothelial growth factor receptor Flt-1 mediates biological activities. Implications for a functional role of placenta growth factor in monocyte activation and chemotaxis. J. Biol. Chem. 271, 17629e17634. Cleaver, O., Melton, D.A., 2003. Endothelial signaling during development (Review). Nat. Med. 9, 661e668. Dayanir, V., Meyer, R.D., Lashkari, K., Rahimi, N., 2001. Identification of tyrosine residues in vascular endothelial growth factor receptor-2/FLK-1 involved in activation of phosphatidylinositol 3-kinase and cell proliferation. J. Biol. Chem. 276, 17686e17692. de Vries, C., Escobedo, J.A., Ueno, H., Houck, K., Ferrara, N., Williams, L.T., 1992. The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science 255, 989e991. Dikov, M.M., Ohm, J.E., Ray, N., Tchekneva, E.E., Burlison, J., Moghanaki, D., Nadaf, S., Carbone, D.F., 2005. Differential roles of vascular endothelial growth factor receptors 1 and 2 in dendritic cell differentiation. J. Immunol. 174, 215e222. Dixelius, J., Makinen, T., Wirzenius, M., Karkkainen, M.J., Wernstedt, C., Alitalo, K., Claesson-Welsh, L., 2003. Ligand-induced vascular endothelial growth factor receptor-3 (VEGFR-3) heterodimerization with VEGFR-2 in primary lymphatic endothelial cells regulates tyrosine phosphorylation sites. J. Biol. Chem. 278, 40973e40979. Eliceiri, B.P., Paul, R., Schwartzberg, P.L., Hood, J.D., Leng, J., Cheresh, D.A., 1999. Selective requirement for Src kinases during VEGF-induced angiogenesis and vascular permeability. Mol. Cell 4, 915e924. Fambrough, D., McClure, K., Kazlauskas, A., Lander, E.S., 1999. Diverse signaling pathways activated by growth factor receptors induce broadly overlapping, rather than independent, sets of genes. Cell 97, 727e741. Folkman, J., 1971. Tumor angiogenesis: therapeutic implications (Review). N. Engl. J. Med. 285, 1182e1186. Fong, G.H., Rossant, J., Gertsenstein, M., Breitman, M.L., 1995. Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 376, 66e70. Fong, G.H., Zhang, L., Bryce, D.M., Peng, J., 1999. Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-1 knock-out mice. Development 126, 3015e3025. Fruttiger, M., 2002. Development of the mouse retinal vasculature: angiogenesis versus vasculogenesis (Review). Invest. Ophthalmol. Vis. Sci. 43, 522e527. Gerber, H.P., McMurtrey, A., Kowalski, J., Yan, M., Keyt, B.A., Dixit, V., Ferrara, N., 1998. Vascular endothelial growth factor regulates endothelial

N. Rahimi / Experimental Eye Research 83 (2006) 1005e1016 cell survival through the phosphatidylinositol 30 -kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J. Biol. Chem. 273, 30336e30343. Gille, H., Kowalski, J., Yu, L., Chen, H., Pisabarro, M.T., Davis-Smyth, T., Ferrara, N., 2000. A repressor sequence in the juxtamembrane domain of Flt-1 (VEGFR-1) constitutively inhibits vascular endothelial growth factor-dependent phosphatidylinositol 30 -kinase activation and endothelial cell migration. EMBO J. 19, 4064e4073. Gragoudas, E.S., Adamis, A.P., Cunningham Jr., E.T., Feinsod, M., Guyer, D.R., 2004. VEGF Inhibition Study in Ocular Neovascularization Clinical Trial Group. Pegaptanib for neovascular age-related macular degeneration. N. Engl. J. Med. 351, 2805e2816. Guba, M., von Breitenbuch, P., Steinbauer, M., Koehl, G., Flegel, S., Hornung, M., Bruns, C.J., Zuelke, C., Farkas, S., Anthuber, M., Jauch, K.W., Geissler, E.R., 2002. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat. Med. 8, 128e135. Guy, P.M., Platko, J.V., Cantley, L.C., Cerione, R.A., Carraway 3rd, K.L., 1994. Insect cell-expressed p180erbB3 possesses an impaired tyrosine kinase activity. Proc. Natl. Acad. Sci. USA 91, 8132e8136. Hanks, S.K., Quinn, A.M., 1991. Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members (Review). Methods Enzymol. 200, 38e62. Hiratsuka, S., Minowa, O., Kuno, J., Noda, T., Shibuya, M., 1998. Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice. Proc. Natl. Acad. Sci. USA 95, 9349e9354. Holmqvist, K., Cross, M.J., Rolny, C., Hagerkvist, R., Rahimi, N., Matsumoto, T., Claesson-Welsh, L., Welsh, M., 2004. The adaptor protein shb binds to tyrosine 1175 in vascular endothelial growth factor (VEGF) receptor-2 and regulates VEGF-dependent cellular migration. J. Biol. Chem. 279, 22267e22275. Huang, J., Kontos, C.D., 2002. PTEN modulates vascular endothelial growth factor-mediated signaling and angiogenic effects. J. Biol. Chem. 277, 10760e10766. Hubbard, S.R., Mohammadi, M., Schlessinger, J., 1998. Autoregulatory mechanisms in protein-tyrosine kinases (Review). J. Biol. Chem. 273, 11987e 11990. Ida, H., Tobe, T., Nambu, H., Matsumura, M., Uyama, M., Campochiaro, P.A., 2003. RPE cells modulate subretinal neovascularization, but do not cause regression in mice with sustained expression of VEGF. Invest. Ophthalmol. Vis. Sci. 44, 5430e5437. Isner, J.M., Ropper, A., Hirst, K., 2001. VEGF gene transfer for diabetic neuropathy. Hum. Gene. Ther. 12, 1593e15104. Jain, R.K., 2003. Molecular regulation of vessel maturation (Review). Nat. Med. 9, 685e693. Jiang, B.H., Zheng, J.Z., Aoki, M., Vogt, P.K., 2000. Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells. Proc. Natl. Acad. Sci. USA 97, 1749e1753. Joukov, V., Kumar, V., Sorsa, T., Arighi, E., Weich, H., Saksela, O., Alitalo, K., 1998. A recombinant mutant vascular endothelial growth factor-C that has lost vascular endothelial growth factor receptor-2 binding, activation, and vascular permeability activities. J. Biol. Chem. 273, 6599e6602. Jussila, L., Alitalo, K., 2000. Vascular growth factors and lymphangiogenesis (Review). Physiol. Rev. 82, 673e700. Kearney, J.B., Ambler, C.A., Monaco, K.A., Johnson, N., Rapoport, R.G., Bautch, V.L., 2002. Vascular endothelial growth factor receptor Flt-1 negatively regulates developmental blood vessel formation by modulating endothelial cell division. Blood 99, 2397e2407. Kearney, J.B., Kappas, N.C., Ellerstrom, C., DiPaola, F.W., Bautch, V.L., 2004. The VEGF receptor flt-1 (VEGFR-1) is a positive modulator of vascular sprout formation and branching morphogenesis. Blood 103, 4527e4535. Kerbel, R., Folkman, J., 2002. Clinical translation of angiogenesis inhibitors (Review). Nat. Rev. Cancer 2, 727e739. Keyt, B.A., Nguyen, H.V., Berleau, L.T., Duarte, C.M., Park, J., Chen, H., Ferrara, N., 1996. Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation of receptor-selective VEGF variants by site-directed mutagenesis. J. Biol. Chem. 271, 5638e5646.

1015

Kim, N.H., Oh, J.H., Seo, J.A., Lee, K.W., Kim, S.G., Choi, K.M., Baik, S.H., Choi, D.S., Kang, Y.S., Han, S.Y., Han, K.H., Ji, Y.H., Cha, D.R., 2005. Vascular endothelial growth factor (VEGF) and soluble VEGF receptor FLT-1 in diabetic nephropathy. Kidney Int. 67, 167e177. Klagsbrun, M., Takashima, S., Mamluk, R., 2002. The role of neuropilin in vascular and tumor biology (Review). Adv. Exp. Med. Biol. 515, 33e48. Klinghoffer, R.A., Sachsenmaier, C., Cooper, J.A., Soriano, P., 1999. Src family kinases are required for integrin but not PDGFR signal transduction. EMBO J. 18, 2459e2471. Lawson, N.D., Mugford, J.W., Diamond, B.A., Weinstein, B.M., 2003. Phospholipase C gamma-1 is required downstream of vascular endothelial growth factor during arterial development. Genes Dev. 17, 1346e1351. Lelievre, E., Bourbon, P.M., Duan, L.J., Nussbaum, R.L., Fong, G.H., 2005. Deficiency in the p110{alpha} subunit of PI 3-kinase results in diminished Tie-2 expression and Tie-2
/
like vascular defects in mice. Blood 105, 3935e3938. Liao, H.J., Kume, T., McKay, C., Xu, M.J., Ihle, J.N., Carpenter, G., 2002. Absence of erythrogenesis and vasculogenesis in Plcg1-deficient mice. J. Biol. Chem. 277, 9335e9341. Liu, M., Regillo, C.D., 2004. A review of treatments for macular degeneration: a synopsis of currently approved treatments and ongoing clinical trials. Curr. Opin. Ophthalmol. 15, 221e226. Matsumoto, T., Bohman, S., Dixelius, J., Berge, T., Dimberg, A., Magnusson, P., Wang, L., Wikner, C., Qi, J.H., Wernstedt, C., Wu, J., Bruheim, S., Mugishima, H., Mukhopadhyay, D., Spurkland, A., Claesson-Welsh, L., 2005. VEGF receptor-2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis. EMBO J. 24, 2342e2353. McTigue, M.A., Wickersham, J.A., Pinko, C., Showalter, R.E., Parast, C.V., Tempczyk-Russell, A., Gehring, M.R., Mroczkowski, B., Kan, C.C., Villafranca, J.E., Appelt, K., 1999. Crystal structure of the kinase domain of human vascular endothelial growth factor receptor 2: a key enzyme in angiogenesis. Structure 7, 319e330. Meyer, R.D., Rahimi, N., 2003. Comparative structure-function analysis of VEGFR-1 and VEGFR-2: What have we learned from chimeric systems? (Review). Ann. N.Y. Acad. Sci. 995, 200e207. Meyer, R.D., Dayanir, V., Majnoun, F., Rahimi, N., 2002. The presence of a single tyrosine residue at the carboxyl domain of vascular endothelial growth factor receptor-2/FLK-1 regulates its autophosphorylation and activation of signaling molecules. J. Biol. Chem. 277, 27081e27087. Meyer, R.D., Latz, C., Rahimi, N., 2003. Recruitment and activation of phospholipase Cgamma1 by vascular endothelial growth factor receptor-2 are required for tubulogenesis and differentiation of endothelial cells. J. Biol. Chem. 278, 16347e16355. Meyer, R.D., Singh, A., Majnoun, F., Latz, C., Lashkari, K., Rahimi, N., 2004a. Substitution of C-terminus of VEGFR-2 with VEGFR-1 promotes VEGFR1 activation and endothelial cell proliferation. Oncogene 23, 5523e5531. Meyer, R.D., Singh, A.J., Rahimi, N., 2004b. The carboxyl terminus controls ligand-dependent activation of VEGFR-2 and its signaling. J. Biol. Chem. 279, 735e742. Meyer, R.D., Mohammadi, M., Rahimi, N., 2006. A single amino acid substitution in the activation loop defines the decoy characteristic of VEGFR-1/ FLT-1. J. Biol. Chem. 281, 867e875. Miller, J.W., Adamis, A.P., Aiello, L.P., 1997. Vascular endothelial growth factor in ocular neovascularization and proliferative diabetic retinopathy (Review). Diabetes Metab. Rev. 13, 37e50. Mori, S., Ronnstrand, L., Yokote, K., Engstrom, A., Courtneidge, S.A., Claesson-Welsh, L., Heldin, C.H., 1993. Identification of two juxtamembrane autophosphorylation sites in the PDGF beta-receptor; involvement in the interaction with Src family tyrosine kinases. EMBO J. 12, 2257e2264. Mossie, K., Jallal, B., Alves, F., Sures, I., Plowman, G.D., Ullrich, A., 1995. Colon carcinoma kinase-4 defines a new subclass of the receptor tyrosine kinase family. Oncogene 11, 2179e2184. Mukhopadhyay, D., Tsiokas, L., Zhou, X.M., Foster, D., Brugge, J.S., Sukhatme, V.P., 1995. Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation. Nature 375, 577e581. Murata, T., Ishibashi, T., Khalil, A., Hata, Y., Yoshikawa, H., Inomata, H., 1995. Vascular endothelial growth factor plays a role in hyperpermeability of diabetic retinal vessels. Ophthalmic Res. 27, 48e52.

1016

N. Rahimi / Experimental Eye Research 83 (2006) 1005e1016

Neagoe, P.E., Lemieux, C., Sirois, M.G., 2005. Vascular endothelial growth factor (VEGF)-A165-induced prostacyclin synthesis requires the activation of VEGF receptor-1 and -2 heterodimer. J. Biol. Chem. 280, 9904e9912. Neri, D., Bicknell, R., 2005. Tumour vascular targeting (Review). Nat. Rev. Cancer 5, 436e446. Neufeld, G., Cohen, T., Gengrinovitch, S., Poltorak, Z., 1999. Vascular endothelial growth factor (VEGF) and its receptors (Review). FASEB J. 13, 9e22. Niu, X.L., Peters, K.G., Kontos, C.D., 2002. Deletion of the carboxyl terminus of Tie2 enhances kinase activity, signaling, and function. Evidence for an autoinhibitory mechanism. J. Biol. Chem. 277, 31768e31773. Odorisio, T., Schietroma, C., Zaccaria, M.L., Cianfarani, F., Tiveron, C., Tatangelo, L., Failla, C.M., Zambruno, G., 2002. Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability. J. Cell Sci. 115, 2559e2567. Pawson, T., 2004. Specificity in signal transduction: from phosphotyrosineSH2 domain interactions to complex cellular systems (Review). Cell 116, 191e203. Pinkas-Kramarski, R., Soussan, L., Waterman, H., Levkowitz, G., Alroy, I., Klapper, L., Lavi, S., Seger, R., Ratzkin, B.J., Sela, M., Yarden, Y., 1996. Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions. EMBO J. 15, 2452e2467. Rahimi, N., 2006. VEGFR-1 and VEGFR-2: two non-identical twins with a unique physiognomy. Front. Biosci. 11, 818e829. Rahimi, N., Kazlauskas, A., 1999. A role for cadherin-5 in regulation of vascular endothelial growth factor receptor 2 activity in endothelial cells. Mol. Biol. Cell 10, 3401e3407. Rahimi, N., Dayanir, V., Lashkari, K., 2000. Receptor chimeras indicate that the vascular endothelial growth factor receptor-1 (VEGFR-1) modulates mitogenic activity of VEGFR-2 in endothelial cells. J. Biol. Chem. 275, 16986e16992. Rameh, L.E., Cantley, L.C., 1999. The role of phosphoinositide 3-kinase lipid products in cell function (Review). J. Biol. Chem. 274, 8347e8350. Reich, S.J., Bennett, J., 2003. Gene therapy for ocular neovascularization: a cure in sight (Review). Curr. Opin. Genet. Dev. 13, 317e322. Robinson, G.S., Ju, M., Shih, S.C., Xu, X., McMahon, G., Caldwell, R.B., Smith, L.E., 2001. Nonvascular role for VEGF: VEGFR-1, 2 activity is critical for neural retinal development. FASEB J. 15, 1215e1217. Rosen, L.S., 2005. VEGF-targeted therapy: therapeutic potential and recent advances (Review). Oncologist 10, 382e391. Saishin, Y., Saishin, Y., Takahashi, K., Lima e Silva, R., Hylton, D., Rudge, J.S., Wiegand, S.J., Campochiaro, P.A., 2003. VEGF-TRAP(R1R2) suppresses choroidal neovascularization and VEGF-induced breakdown of the blood-retinal barrier. J. Cell Physiol. 195, 241e248. Sakurai, Y., Ohgimoto, K., Kataoka, Y., Yoshida, N., Shibuya, M., 2005. Essential role of Flk-1 (VEGF receptor 2) tyrosine residue 1173 in vasculogenesis in mice. Proc. Natl. Acad. Sci. USA 102, 1076e1081. Schlessinger, J., 2000. Cell signaling by receptor tyrosine kinases. Cell 103, 211e215. Schlessinger, J., 2003. Signal transduction. Autoinhibition control. Science 300, 750e752. Schwesinger, C., Yee, C., Rohan, R.M., Joussen, A.M., Fernandez, A., Meyer, T.N., Poulaki, V., Ma, J.J., Redmond, T.M., Liu, S., Adamis, A.P., D’Amato, R.J., 2001. Intrachoroidal neovascularization in transgenic mice overexpressing vascular endothelial growth factor in the retinal pigment epithelium. Am. J. Pathol. 158, 1161e1172. Sekiya, F., Bae, Y.S., Rhee, S.G., 1999. Regulation of phospholipase C isozymes: activation of phospholipase C-gamma in the absence of tyrosinephosphorylation (Review). Chem. Phys. Lipids. 98, 3e11. Seye, C.I., Yu, N., Gonzalez, F.A., Erb, L., Weisman, G.A., 2004. The P2Y2 nucleotide receptor mediates vascular cell adhesion molecule-1 expression through interaction with VEGF receptor-2 (KDR/Flk-1). J. Biol. Chem. 279, 35679e35686. Shih, S.C., Ju, M., Liu, N., Smith, L.E., 2003. Selective stimulation of VEGFR-1 prevents oxygen-induced retinal vascular degeneration in retinopathy of prematurity. J. Clin. Invest. 112, 50e57. Singh, A.J., Meyer, R.D., Band, H., Rahimi, N., 2005. The carboxyl terminus of VEGFR-2 is required for PKC-mediated down-regulation. Mol. Biol. Cell 16, 2106e2118.

Smith, L.E., 2004. Pathogenesis of retinopathy of prematurity. Growth Horm. IGF Res. 14 (Suppl. A), S140eS144. Soldi, R., Mitola, S., Strasly, M., Defilippi, P., Tarone, G., Bussolino, F., 1999. Role of alphavbeta3 integrin in the activation of vascular endothelial growth factor receptor-2. EMBO J. 18, 882e892. Stacker, S.A., Vitali, A., Caesar, C., Domagala, T., Groenen, L.C., Nice, E., Achen, M.G., Wilks, A.F., 1999. A mutant form of vascular endothelial growth factor (VEGF) that lacks VEGF receptor-2 activation retains the ability to induce vascular permeability. J. Biol. Chem. 274, 34884e34892. Sudhakar, A., Sugimoto, H., Yang, C., Lively, J., Zeisberg, M., Kalluri, R., 2003. Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by alpha v beta 3 and alpha 5 beta 1 integrins. Proc. Natl. Acad. Sci. USA 100, 4766e4771. Takahashi, T., Yamaguchi, S., Chida, K., Shibuya, M., 2001. A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-A-dependent activation of PLC-gamma and DNA synthesis in vascular endothelial cells. EMBO J. 20, 2768e2778. Tammela, T., Enholm, B., Alitalo, K., Paavonen, K., 2005. The biology of vascular endothelial growth factors (Review). Cardiovasc. Res. 65, 550e563. Thakker, G.D., Hajjar, D.P., Muller, W.A., Rosengart, T.K., 1999. The role of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling. J. Biol. Chem. 274, 10002e10007. Till, J.H., Becerra, M., Watty, A., Lu, Y., Ma, Y., Neubert, T.A., Burden, S.J., Hubbard, S.R., 2002. Crystal structure of the MuSK tyrosine kinase: insights into receptor autoregulation. Structure (Camb.) 10, 1187e1196. Waltenberger, J., Claesson-Welsh, L., Siegbahn, A., Shibuya, M., Heldin, C.H., 1994. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J. Biol. Chem. 269, 26988e26995. Wegewitz, U., Gohring, I., Spranger, J., 2005. Novel approaches in the treatment of angiogenic eye disease (Review). Curr. Pharm. Des. 11, 2311e2330. Wey, J.S., Fan, F., Gray, M.J., Bauer, T.W., McCarty, M.F., Somcio, R., Liu, W., Evans, D.B., Wu, Y., Hicklin, D.J., Ellis, L.M., 2005. Vascular endothelial growth factor receptor-1 promotes migration and invasion in pancreatic carcinoma cell lines. Cancer 104, 427e438. Wiesmann, C., Fuh, G., Christinger, H.W., Eigenbrot, C., Wells, J.A., de Vos, A.M., 1997. Crystal structure at 1.7 A resolution of VEGF in complex with domain 2 of the Flt-1 receptor. Cell 91, 695e704. Witmer, A.N., Vrensen, G.F., Van Noorden, C.J., Schlingemann, R.O., 2003. Vascular endothelial growth factors and angiogenesis in eye disease (Review). Prog. Retin. Eye Res. 22, 1e29. Wybenga-Groot, L.E., Baskin, B., Ong, S.H., Tong, J., Pawson, T., Sicheri, F., 2001. Structural basis for autoinhibition of the Ephb2 receptor tyrosine kinase by the unphosphorylated juxtamembrane region. Cell 106, 745e757. Yamazaki, T., Akada, T., Niizeki, O., Suzuki, T., Miyashita, H., Sato, Y., 2005. Puromycin-insensitive leucyl-specific aminopeptidase (PILSAP) binds and catalyzes PDK1, allowing VEGF-stimulated activation of S6K for endothelial cell proliferation and angiogenesis. Blood 104, 2345e2352. Yancopoulos, G.D., Davis, S., Gale, N.W., Rudge, J.S., Wiegand, S.J., Holash, J., 2000. Vascular-specific growth factors and blood vessel formation (Review). Nature 407, 242e248. Yeynolds, A.R., Reynolds, L.E., Nagel, T.E., Lively, J.C., Robinson, S.D., Hicklin, D.J., Bodary, S.C., Hodivala-Dilke, K.M., 2004. Elevated Flk1 (vascular endothelial growth factor receptor 2) signaling mediates enhanced angiogenesis in beta3-integrin-deficient mice. Cancer Res. 64, 8643e8650. Yoshikawa, S., Bonkowsky, J.L., Kokel, M., Shyn, S., Thomas, J.B., 2001. The derailed guidance receptor does not require kinase activity in vivo. J. Neurosci. 21, RC119eRC121. Zakarija, A., Soff, G., 2005. Update on angiogenesis inhibitors (Review). Curr. Opin. Oncol. 17, 578e583. Zarbin, M.A., 2004. Current concepts in the pathogenesis of age-related macular degeneration. Arch. Ophthalmol. 122, 598e614. Zeng, H., Dvorak, H.F., Mukhopadhyay, D., 2001. Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) peceptor-1 downmodulates VPF/VEGF receptor-2-mediated endothelial cell proliferation, but not migration, through phosphatidylinositol 3-kinase-dependent pathways. J. Biol. Chem. 276, 26969e26979.